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ASH2011 poster final .pdf


Original filename: ASH2011 poster-final.pdf
Title: ASH2011 poster-final
Author: padrone

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GM-CSF signaling abnormalities in Chronic Myelomonocytic
Leukemia
Eric Padron, Jeffrey S. Painter, Adam W Mailloux, Jessica M. McDaniel,
McDaniel, Christopher Bebbington, Mark Braer,
Braer,
Rami S. Komrokji, Alan F. List, and Pearlie K EplingEpling-Burnette
Abstract

Results

•Background: Chronic Myelomonocytic Leukemia (CMML) and Juvenile Myelomonocytic leukemia (JMML)
are classified as MDS/MPN in the WHO classification system. Despite sharing clinical and histological
features, CMML is characterized by a heterogeneous collection of molecular lesions while JMML is defined by
well-established molecular aberrations clustered along the RAS pathway leading directly to GM-CSF
hypersensitivity; a pathognomonic characteristic of JMML. Here we test whether a molecular signature for
GM-CSF hypersensitivity in JMML, determined by the pSTAT5 activation assay, is also present in CMML and
whether this signature clusters within a specific CMML subgroup.
•Methods: Cryopreserved bone marrow aspirates from 24 patients with newly diagnosed or relapsed CMML
were obtained from the Moffitt Cancer Center Tissue Repository. Cells were thawed and rested in Stem Span
H3000 with 10% FBS for 2 hours and then either starved for one hour in serum-free media, serum free group
(n=12), or rested in Stem Span for an additional hour, serum group (n=12), prior to stimulation with G-CSF, IL3, or GM-CSF for 15 minutes and then fixed and permeabilized with formaldehyde and methanol, as previously
described. Samples were stained with an anti-pSTAT5(Y-694) antibody and analyzed by flow cytometry
(Kotecha, Cancer Cell. 2009). Cells stained with isotype-control antibody were used to establish the threshold
for basal STAT5 phosphorylation. Because STAT5 was constitutively phosphorylated in serum, and to a lesser
extent in serum-free conditions, inducible cytokine activation was defined as the percentage of pSTAT5
positive cells above untreated samples in both CMML and healthy controls. A retrospective chart review was
performed to obtain clinical variables including age, sex, WHO classification, Dusseldorf scoring system, MD
Anderson scoring system, WBC, peripheral monocyte count, blast percentage, anemia, platelet count,
splenomegaly, and metaphase cytogenetics.
•Results: The percentage of pSTAT5 responsive cells after G-CSF stimulation with doses up to 10 ng/ml was
similar in cases and normal BM controls (p=0.14), whereas, a statistically significant increase in the percentage
of inducible pSTAT5 positive cells was observed with GM-CSF 0.1 ng/ml (p=0.04), GM-CSF 1 ng/ml
(p=0.02), and GM-CSF 10 ng/ml (p=0.01) in CMML BM cells compared to healthy donor BM cells, as shown
in Figure 1. Using one standard deviation below the mean as a cut point, only 5 patients failed to show GMCSF hypersensitivity in the serum (n=3) and serum-free groups (n=2), respectively. IL-3 and GM-CSF play
similar roles in hematopoietic growth through the activation of JAK2/STAT5 and share a common beta-chain
required for signaling. Signaling mediated by GM-CSF and IL3 converge to activate RAS and other
downstream intermediates that regulate DNA synthesis, cell-cycle progression and suppression of
apoptosis. The concentration of IL3 required to induce STAT5 phosphorylation was 10-fold greater than GMCSF in CMML cells, but the percentage of cells responsive to IL3 was greater in CMML cases compared to
controls at 10 ng/ml (p=0.02). Analysis of the percentage of GM-CSF hypersensitive cells and clinical
parameters revealed no associations with age at onset, WHO classification, Dusseldorf scoring system, MD
Anderson scoring system, blast percentage, anemia, platelet count, splenomegaly, or karyotype. The percentage
of pSTAT5 positive cells with GM-CSF 0.1 ng/ml positively correlated with the total leukocyte (p=0.03) and
total monocyte (p=0.02) count indicating that the JAK2/STAT5 signaling response is indicative of disease
burden.
•Conclusions: Based on the threshold for cytokine stimulation and percentage of cells that display pSTAT5
induction, CMML appears to preferentially utilize GM-CSF for survival and/or expansion. Although RAS
mutations were not assessed, CMML cells were preferentially sensitive to GM-CSF in newly diagnosed cases
independent of cytogenetic abnormalities suggesting that JMML and CMML share biological features of GMCSF hypersensitivity

GM-CSF dependent pSTAT5 induction in CMML patients versus controls. Frozen, whole bone
marrow aspirates of patient samples were thawed in Stemspan H3000 containing serum and
then rested in serum free RPMI for one hour. GM-CSF was added and cells were fixed and
permeabilized after 15 minutes of stimulation. Cells were stained with an Alexa647 conjugated
anti-pY647STAT5 antibody and analyzed on an LSR II FACS. This is a representative CMML
and normal frozen sample.

A.

Percent pSTAT5 positive cells of the 12 CMML samples at all cytokine doses in serum conditions.
Compared to normal controls, 70% of CMML cells are hyper-responsive to GM-CSF at all doses
except 0.01ng/ml and are hyper-responsive to IL3 at 10ng/ml. Hyper-responsiveness is defined as those
CMML samples that exhibit pSTAT5 induction at one standard deviation above controls. (A)
Percentage of CMML cells with positive pSTAT5. (B) Percentage of normal control cells with positive
pSTAT5.

B.

Basal measurement of pSTAT5 in CMML patients versus controls. Frozen CMML samples were
prepared as previously described. Cells were fixed, permeabilized, and stained with an antipY647STAT5 antibody and analyzed by FACS. (A) Representative CMML and control samples in
serum and serum free conditions. (B) Basal pSTAT5 levels of 20 CMML patient samples and 6 normal
controls.

pSTAT5 induction of the entire CMML cohort (20 patients) at all cytokine doses in serum free conditions. Compared
to normal controls (6), all CMML cells are hyper-responsive to GM-CSF at all doses except 0.01ng/ml and are hyperresponsive to IL3 at 10ng/ml. Hyper-responsiveness is defined as those CMML samples that exhibit pSTAT5
induction at one standard deviation above controls. (A) Percentage of CMML cells with positive pSTAT5. (B)
Percentage of normal control cells with positive pSTAT5. (C) Mean Fluorescent Intensity (MFI) of pSTAT5 in
CMML cells. (D) MFI of pSTAT5 in normal controls.

Patient Characteristics
Clinical parameters outline in the abstract were statistically tested by Mann-Whitney (categorical
values) or Spearman (continuous values) for correlation to p-STAT5. Only WBC (A. and B.) and
peripheral monocyte counts (C. and D.) correlated to percent positive and MFI pSTAT5. A linear
regression with 95% confidence intervals (red) are noted. Twenty patients were tested in serum free
conditions.

Conclusion
• CMML primary cells display hyper-responsive GM-CSF dependent pSTAT5 induction in vitro .
• CMML primary cells have increased basal pSTAT5 levels compared to controls .
• CMML primary cells are selectively sensitive to low dose GM-CSF but display unselected hyperresponsiveness at high doses.
• Future studies are necessary to determine whether pSTAT5 responsiveness is important to CMML
survival in vitro and in vivo.

Acknowledgements
Cytokine dependent pSTAT5 induction in CMML patients versus controls. Frozen, whole bone marrow aspirates of
patient samples prepared as previously described. Cytokines were added and cells were fixed and permeabilized after
15 minutes of stimulation. Cells were stained with an Alexa647 conjugated anti-pY647STAT5 antibody and
analyzed on an LSR II FACS. This is a representative CMML and normal frozen sample.

•This work was funded by KaloBios Pharmaceuticals
•All primary cells were acquired from the Moffitt Cancer Center tissue core (TCC)
•Flow cytometry was performed using the Moffitt Cancer Center Flow Cytometry Core


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